Catalsimeter with time measuring circuitry for determining reactant concentration level

ABSTRACT

This invention relates to an apparatus and a method for determining the concentration of a first reactant in a medium. A disc is saturated with a solution containing the first reactant and immersed into a second reactant fluid. A chemical reaction occurs between the first and second reactants to produce a gas which is trapped by the disc. The disc is buoyed to the surface of the second reactant fluid by the gas produced. The process is electronically timed using photocells to start and stop an electronic clock and a digital display displays the time elapsed in tenths of seconds.

The present application is a division of application Ser. No. 487,615,filed July 11, 1974, and now U.S. Pat. No. 3,948,730, issued on Apr. 6,1976.

INTRODUCTION AND GENERAL DISCUSSION

This invention relates to an apparatus and a method for determining theconcentration of a first reactant in a medium. In particular, thepresent invention provides a method and apparatus for determining theconcentration of a catalase enzyme in a medium.

In the determination of the concentration of a first reactant in amedium, the medium can be immersed into a fluid containing a secondreactant in a known concentration which reacts with the first reactantto produce a gas. If the chemical composition of the reacting fluid isknown, then the rate of gas production can be used to calculate thedesired concentration of first reactant using, for example, theGagnon-Hunting method. This rate can be measured electronically if agas-trapping object of a known size is saturated with the mediumcontaining the first reactant and then immersed into the fluidcontaining the second reactant. The gas trapped by the gas-trappingobject will reduce the weight of the gas-trapping object by theresultant bouyant force, and the rate of increase of the bouyant forceis proportional to the rate of gas production.

If the gas-trapping object sinks in the fluid when not filled with thegas to be produced, and also rises to the surface of the fluid whenpartially filled with the trapped gas produced, the time taken for theobject to sink and rise to the surface of the fluid can be measuredelectronically. This time will be proportional to the buoyant forceacting on the gas-trapping object, and hence proportional to the rate ofgas production in the reaction, and therefore proportional to theconcentration of the first reactant in the medium.

In the particular case where the concentration of the catalase enzyme isto be determined, the second reactant is H₂ O₂. H₂ O₂ is typicallyplaced in the fluid at a required concentration by volume. Thegas-trapping object can be conveniently a disc shaped piece of filterpaper of a known size.

Occasionally, when the disc of filter paper is dropped into the H₂ O₂,it does not immediately sink but flips over and rises to the surface ofthe fluid almost immediately after it is dropped. This action causeslight beams from the photocell detector to be broken twice when the discenters the fluid, hence starting the clock and stopping it immediatelyafterwards. This situation was corrected by an appropriate controlcircuit which will ignore a signal from the photodetector unless thatsignal is received after a predetermined time has elapsed. This allowsthe disc to flip over without affecting the electronic clock.

STATEMENT OF INVENTION

In accordance with the present invention, there is provided a method ofmeasuring the concentration of a first reactant in a medium by reactingit with a second reactant fluid of a predetermined concentration toproduce a gas, said method comprising the steps of:

a. saturating a gas-trapping object of known weight with said mediumcontaining said first reactant;

b. placing said gas-trapping object into an open container whichcontains, to a predetermined depth, a quantity of said fluid; and

c. measuring electronically the time taken for said gas-trapping objectto sink into said fluid, and the time taken to produce a quantity of gassufficient to buoy said gas-trapping object to the surface of saidfluid, wherein the time taken for said gas-trapping object to sink intosaid fluid and to be buoyed to the surface by said gas is proportionalto the concentration of said first reactant in said medium.

In accordance with the present invention, there is also provided acircuit for electronically measuring and displaying the time taken for agas-trapping object saturated with a medium containing a first reactantto sink and rise to the surface of a fluid containing a second reactantat a predetermined concentration in a container due to the reaction ofsaid first and second reactants, said circuit comprising:

a. photo-detector means for producing a start pulse when saidgas-trapping object enters said fluid and a stop pulse when saidgas-trapping object rises to the surface of said fluid; and

b. clock means operatively associated with said photo-detector means fordisplaying elapsed time between the receipt of said start pulse and saidstop pulse, said elapsed time being proportional to the concentration ofsaid first reactant in said medium.

DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the accompanyingdrawings, in which:

FIG. 1 is a side elevation of a part of a particular embodimentaccording to the present invention;

FIG. 2 is a top plan view of that part of the embodiment shown in FIG.1;

FIG. 3 is a block diagram of an embodiment in accordance with thepresent invention; and

FIG. 4 is a schematic diagram of an embodiment in accordance with thepresent invention.

DETAILED DESCRIPTION

Referring to FIGS. 1 and 2, the apparatus shown is comprised of lamps 10and 11 affixed to and oriented 90° about a hollow container 30. Lamps 10and 11 provide orthogonally oriented light beams 12 and 14 which impingeon photoresistors 20 and 21 respectively. These photoresistors areattached to opposite sides of the container from the lamps. Normally,test tube 40 will rest in the container on test-tube stopper 41 and willbe held in place by test tube spring 42 which encircles the test tube.When in use, test tube 40 will be partially filled with a reactant fluid16 of known chemical composition. The height of the fluid in the testtube is such as to provide a surface 18 which will correspond to a planedefined by the light beams which enter the photoresistors, so that anyobject entering or floating on the surface of the fluid will interruptone or both of the light beams. Two orthogonally oriented lamp andphotoresistor combinations are used to ensure that at least one of thebeams 12 or 14 will be interrupted regardless of the position that theobject enters the fluid or floats on the surface 18 of the fluid 16.

A gas-trapping element 19 (FIG. 1), for example a disc of filter paperof known dimension, is immersed in a fluid medium containing the firstreactant, the concentration of which is to be determined. The disc, soconditioned, is then dropped into the test tube 40, breaking one or bothof the light beams 12 and 14 thereby starting an electronic counterwhich will be described in detail below, and sinks into the fluidcolumn. The first reactant reacts with the reactant fluid 16 to producea gas. The disc traps the gas so produced, becoming buoyant andeventually floating to the surface 18, breaking one or both of the lightbeams 12 and 14, thereby stopping the counter. The timed reading of thecounter will be proportional to the concentration of the first reactantin the medium.

FIG. 3 is a block diagram of an electronic circuit which is adapted tomeasure the time elapsed while the gas-trapping object is immersed inthe fluid. Photodetector 50 consists of lamps 10 and 11 andphotoresistors 20 and 21 as shown in FIGS. 1 and 2 in a circuit such asthat depicted in FIG. 4. When either light beam is interrupted, as wouldhappen if an object were to enter the fluid or float on its surface, apulse is fed from photodetector circuit 50 to the pulse shaper 51. Pulseshaper 51 merely modifies the pulse fed from the photodetector circuit50 in that the output of pulse shaper 51 consists of square pulses ofeither the same or reverse polarity as the input pulses. These squarepulses are fed into control circuit 52 which starts or stops a counter54 and optionally activates warning device 60. Control circuit 52 feedsa modified square pulse from that received from pulse shaper 51. Thepulse is of the appropriate polarity to start or stop counter 54, andthe pulse is of sufficient length so that if the gas-trapping objectflips over and interrupts the light beam more than once before sinking,the second pulse generated by the pulse shaper will be ignored. Thelength of the output pulse from control circuit 52 is easily adjusted.Optional warning device 60 could consist of a sound or light emittingmechanism to either operate while the counter is activated orimmediately when the gas trapping object floats to the surface of thefluid.

Clock pulse shaper 53 merely changes the 60 Hertz alternating linevoltage into a 60 cycle square wave, and feeds these square wave pulsesto counter 54. Counter 54 receives an input from control circuit 52which either starts or stops its counting. Counter 54 is designed tooutput every 6th pulse to display 56. Hence, while counter 54 isoperating, display 56 digitally records pulses received at intervals of1/10 of a second. Since counter 54 operates only while the gas-trappingobject is immersed in the fluid, display 56 digitally displays theinterval of time elapsed from the time that the object enters the fluiduntil it floats to the surface of the fluid. Display 56 is anappropriate integrated circuit capable of displaying digitally theoutput of counter 54. Reset circuit 55 consists of a push button which,when depressed, automatically resets counter 54 and display 56 to zero.

Referring now to FIG. 4, photodetector circuit 50 could be implementedas shown. While the light beam is uninterrupted, the transistor isbiased into saturation so that the input to pulse shaper circuit 51 isat a reference potential. In the embodiment shown in FIG. 4, thereference potential will be of a positive polarity and the remainingcircuitry is adapted to be compatible with this positive referencepolarity. However, by changing the transistor type, a referencepotential of the opposite polarity could be derived. It should beunderstood that the scope of the present invention contemplates bothsituations. When an object interrupts either light beam, thephotoresistors change in value so as to bias the transistor into cutoffthus producing a negative going pulse which is fed to the pulse shaper51. Photodetector circuit 50 contains an anti-heat drift circuit tocompensate for voltage changes due to heat caused by the lamps. Pulseshaper 51 is an integrated circuit which merely changes the input pulsefrom photodetector circuit 50 to a well defined square pulse ofappropriate polarity to activate control circuit 52. A possibleintegrated circuit for the pulse shaper 51 is a Schmidt Trigger No.SN5413N manufactured by Texas Instruments.

Control circuit 52 could be implemented as shown in FIG. 4. This circuitconsists of a NOR gate 70 and a monostable multivibrator 72. When anobject breaks one or both light beams 12 or 14 as shown in FIG. 2, a lowlevel logic pulse is fed from pulse shaper 51 to one input of gate 70.If monostable multivibrator 72 is in its stable state, a low level logicvoltage is applied to the second input of gate 70. This low level logicvoltage will ready gate 70 only if monostable multivibrator 72 is readyto receive a pulse from gate 70. If both inputs of gate 70 areconditioned with low level logic voltages, gate 70 produces a high levellogic voltage which is fed to monostable multivibrator 72 which changesstate. Monostable multivibrator 72 changes state for a predeterminedtime period depending on the value of capacitor Cl and resistor Rl. Thistime period is made long enough to filter out any additional pulsescoming from the pulse shaper 51 due to the gas-trapping object flippingover upon entry into the fluid.

Counter 54 is composed of an integrated circuit, such as Type SN7492Amanufactured by Texas Instruments, and NOR gates 73 and 74, as shown inFIG. 4. When the light beam is interrupted, a low pulse is fed fromcontrol circuit 52 to activate counter 54. Substantially square clockpulses having a 60 cycle repetition rate are also fed to counter 54 fromclock pulse shaper 53. When activated, counter 54 feeds every sixthpulse to display 56, thus counting in tenths of seconds. Invertor gate73 assures that the counter will count the clock pulses only whenactivated, since the output from NOR gate 74 is high when the counter isnot activated by control circuit 52 and low when activated. Clock pulseshaper 53 merely changes the 60 hertz line voltage into 60 hertz squarewave pulses. A typical device for this purpose would be a SchmidtTrigger No. SN7413N, manufactured by Texas Instruments. Reset circuit 55is comprised on invertor gate 76 and push button 78. When depressed,push buttons 78 resets counter 45 and display 56 to zero. Display 56numerically displays the number of pulses received from counter 54. Thismeans it displays the reaction time in tenths of seconds. A typicaldisplay would be type TlL306, manufactured by Texas Instruments.

The embodiment described above is designed to operate on a 60 hertz linefrequency. It should be understood that if a different line frequency isused, the counter 54 would be changed accordingly. For example, if a 50cycle line frequency was used the counter would be changed to a divideby 5 counter.

What we claim as our invention is:
 1. A circuit for electronicallymeasuring and displaying a readout of elapsed time for a gas-trappingobject saturated with a medium containing a first reactant to buoyantlyrise within and break the surface of a fluid containing a secondreactant at a predetermined concentration in a container due to thereaction of said first and second reactants to produce a gas followingsaid gas-trapping object breaking said surface and sinking in the fluid,said circuit comprising:a. photo-detector means for producing a startpulse when said gas-trapping object breaks said surface to sink in saidfluid and a stop pulse when said gas-trapping object buoyantly rises andagain breaks the surface of said fluid; b. clock means for displayingsaid elapsed time between said start pulse and said stop pulse, saidelapsed time being proportional to the concentration of said firstreactant in said medium; and c. means for electrically connecting saidclock means and said photo-detector means, said electrically connectingmeans including means for passing said stop pulse only if said stoppulse should be produced after a predetermined time period followingsaid production of said start pulse.
 2. A circuit for electronicallymeasuring and displaying a readout of elapsed time for a gas-trappingobject saturated with a medium containing a first reactant to buoyantlyrise within and break the plane of the surface of a fluid containing asecond reactant at a predetermined concentration in a container due tothe reaction of said first and second reactants to produce a gasfollowing said gas-trapping object having broken said plane and sunkenin the fluid, said circuit comprising:a. photo-detector means forproducing a start pulse when said gas-trapping object breaks said planeto sink in said fluid and a stop pulse when said gas-trapping objectbuoyantly rises and again breaks said plane of said fluid; b. clockmeans for displaying said elapsed time between said start pulse and saidstop pulse, said elapsed time being of at least a predetermined durationthereby to be proportional to the concentration of said first reactantin said medium; and, c. means for electrically connecting said clockmeans and said photo-detector means.
 3. A circuit according to claim 2wherein said photo-detector means includes at least one light source andphoto-resistor, said light source and photo-resistor being paired andarranged on opposite sides of said container in a plane of the surfaceof said fluid.
 4. A circuit according to claim 2 wherein saidphoto-detector means includes two light sources and photo-resistors,each said light source and photo-resistor being paired and arranged onopposite sides of said container in a plane of the surface of saidfluid, said pairs being substantially orthogonally positioned withrespect to one another.
 5. A circuit according to claim 4 wherein saidphoto-detector means includes switch means, and said photo-resistors ofsaid light source-photo-resistor pairs connected to said switch means,said switch means producing a pulse each time said gas-trapping objectpasses between at least one of said light source-photo-resistor pairs.6. A circuit for electronically measuring and displaying a readout ofelapsed time for a gas-trapping object saturated with a mediumcontaining a first reactant to buoyantly rise within and break thesurface of a fluid containing a second reactant at a predeterminedconcentration in a container due to the reaction of said first andsecond reactants to produce a gas following said gas-trapping objectbreaking said surface and sinking in the fluid, said circuitcomprising:a. photo-detector means for producing a start pulse when saidgas-trapping object breaks said surface to sink in said fluid and a stoppulse when said gas-trapping object buoyantly rises and again breaks thesurface of said fluid, said photo-detector means including1. two lightsources,
 2. two photo-resistors, each said light source andphoto-resistor being paired and arranged on opposite sides of saidcontainer in a plane of the surface of said fluid, said pairs beingsubstantially orthogonally positioned with respect to one another, and3. switch means, said photo-resistors of said lightsource-photo-resistor pairs connected to said switch means, said switchmeans producing a pulse each time said gas-trapping object passesbetween at least one of said light source-photo-resistor pairs; b. clockmeans for displaying said elapsed time between said start pulse and saidstop pulse, said elapsed time being proportional to the concentration ofsaid first reactant in said medium; and, c. means for electricallyconnecting said clock means and said photo-detector means, said meansincluding1. pulse shaper means, said pulse shaper means producing asubstantially square output pulse upon receipt of each said pulse fromsaid switch means, and
 2. means connecting said pulse shaper means andsaid switch means.
 7. A circuit according to claim 6 wherein saidelectrical connecting means further includes control circuit meansconnected to said pulse shaper, said control circuit producing saidstart pulse upon receipt of said substantially square output pulse andthereafter said stop pulse if and only if said control circuit meansreceives said next substantially square output pulse a predeterminedtime period after receipt of the preceding substantially square outputpulse.
 8. A circuit according to claim 7 wherein said clock meansincludes clock pulse shaper means, said clock pulse shaper meansconnected to means providing a substantially sinusoidal 60 hertz outputsignal for producing substantially square clock pulses having a 60 cyclerepetition rate.
 9. A circuit according to claim 8 wherein said clockpulse shaper means is a Schmidt trigger.
 10. A circuit according toclaim 8 wherein said clock means further includes electronic countingmeans, said electronic counting means being connected to said controlcircuit means and to said clock pulse shaper means, said electroniccounting means starting its count upon receipt of said start pulse,stopping its count upon receipt of said stop pulse and, while countingsaid clock pulses, producing a count output pulse upon counting eachsixth clock pulse, thereby producing count output pulses at intervals of0.1 seconds.
 11. A circuit according to claim 10 wherein said clockmeans further includes display means, said display means connected tosaid electronic counting means for displaying the count of the number ofsaid count output pulses.
 12. A circuit according to claim 11 whereinsaid display means is a light emitting diode display.